Expression of Rgmc, the murine ortholog of

RED CELLS
Brief report
Expression of Rgmc, the murine ortholog of hemojuvelin gene, is modulated
by development and inflammation, but not by iron status or erythropoietin
Jan Krijt, Martin Vokurka, Ko-Tung Chang, and Emanuel Nečas
Mutations of hepcidin (HAMP) and hemojuvelin (HJV) genes have been recently
demonstrated to result in juvenile hemochromatosis. Expression of HAMP is regulated by iron status or infection, whereas
regulation of HJV is yet unknown. Using
quantitative real-time polymerase chain
reaction, we compared expression of
Hamp and Rgmc (the murine ortholog of
HJV) in livers of mice treated with iron,
erythropoietin, or lipopolysaccharide
(LPS), as well as during fetal and postnatal development. Iron overload increased
Hamp expression without effect on Rgmc
mRNA. Erythropoietin decreased Hamp
mRNA, but Rgmc expression was unchanged. Hamp mRNA level decreased
after birth by 4 orders of magnitude, without significant changes in Rgmc expression. Administration of LPS elevated
Hamp mRNA levels, while markedly decreasing hepatic Rgmc mRNA levels (to
⬃5% after 6 hours). The responses of
Hamp and Rgmc were quite different and
suggested that human HJV expression
could be modulated by inflammation.
(Blood. 2004;104:4308-4310)
© 2004 by The American Society of Hematology
Introduction
During the past few years, a number of new genes participating in
iron metabolism have been identified. Mutations in 2 genes,
hepcidin (HAMP)1 and hemojuvelin (HJV)2,3 have been shown to
result in juvenile hemochromatosis. Hepcidin, a small peptide
synthesized predominantly in hepatocytes, is emerging as an
important regulator of iron homeostasis, which inhibits iron
absorption from the intestine and iron release from macrophages.
Hepcidin expression is controlled by iron status and erythropoietic
activity, as well as by inflammatory stimuli4; inappropriate expression of hepcidin probably plays a role in the pathophysiology of
hereditary hemochromatosis and anemia of inflammation.5 On the
other hand, the function and regulation of hemojuvelin are at
present unknown. Prior to identification of the HJV gene, it was
speculated that its product could function in the hepcidin
signaling pathway, possibly as a hepcidin receptor,5 whereas a
current concept proposes that hemojuvelin could modulate
hepcidin expression.2,6
Orthologs of the HJV gene have been identified in zebrafish,
mice, and rats2; the mouse HJV ortholog Rgmc is, like HJV,
expressed mainly in skeletal muscle, heart, and liver.7 The aim of
the present study was to examine whether experimental conditions
known to influence hepatic Hamp expression in mice will also
change hepatic Rgmc mRNA levels and to compare possible
similarities or discrepancies in the regulation of these 2 genes.
Study design
All animal experiments were approved by the Animal Care Committee of
the First Faculty of Medicine. Male C57BL/6N mice (Charles River,
From the Institute of Pathological Physiology, First Faculty of Medicine, Charles
University, Prague, Czech Republic.
Submitted June 28, 2004; accepted August 1, 2004. Prepublished online as
Blood First Edition Paper, August 17, 2004; DOI 10.1182/blood-2004-06-2422.
Supported by grant VZ 111100003 from the Ministry of Education of the Czech
Republic. J.K. and M.V. contributed equally to this study.
4308
Sulzfeld, Germany) were treated with lipopolysaccharide (LPS, serotype
0111:B4, 1 mg/kg intraperitoneally; Sigma Aldrich, Prague, Czech Republic) and humanely killed by cervical dislocation after 90 minutes or 6 hours.
Iron overload (600 mg/kg) was induced by a single subcutaneous injection
of iron polyisomaltosate (Ferrum Lek; Lek, Ljubljana, Slovenia); mice
were humanely killed 1 week or 3 weeks after application. Erythropoietin
(EPREX 10 000, Cilag AG, Schaffhausen, Switzerland) was administered
at 50 U/mouse for 4 days, and mice were killed on day 5.
Liver RNA was extracted using RNABlue (Top-Bio, Prague, Czech
Republic), treated with DNase I (Gibco, Life Technologies, Gaithersburg,
MD), and 1 ␮g total RNA was reverse transcribed by the RevertAid
First-Strand cDNA synthesis kit (Fermentas, Vilnius, Lithuania).
Levels of Hamp and Rgmc mRNA were determined by real-time
polymerase chain reaction (PCR) on a Roche LightCycler instrument, using
LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics,
Mannheim, Germany). Primer sequences were: ␤-actin forward 5⬘GACATGGAGAAGATCTGGCA-3⬘, reverse 5⬘-GGTCTTTACGGATGTCAACG-3⬘; Hamp forward 5⬘-CTGAGCAGCACCACCTATCTC-3⬘, Hamp
reverse 5⬘-TGGCTCTAGGCTATGTTTTGC-3⬘; Rgmc forward 5-CCCAGATCCCTGTGACTATGA -3, Rgmc reverse 5-CAGGAAGATTGTCCACCTCAG -3. Rgmc primers were designed to amplify a sequence from exons
3 and 4 of Rgmc DNA.2 Because 5 possible splice variants have been
reported for human HJV,2 data from representative experiments were
verified with 2 alternative primer pairs targeting sequences from exon 1 and
exon 4, respectively.
Target mRNA amounts were normalized to ␤-actin mRNA, and
calculated as described previously,8 assuming exact doubling of amplified cDNA in each PCR cycle. Results are expressed as the relative
amount of target mRNA in comparison to ␤-actin mRNA (Figure 1), or
as percent of ␤-actin–normalized target mRNA in experimental groups
versus control groups (Table 1). Statistical analyses were performed
using the 2-tailed t test.
Reprints: Jan Krijt, Institute of Pathological Physiology, First Faculty of
Medicine, Charles University, U nemocnice 5, 128 53 Prague, Czech Republic;
e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
BLOOD, 15 DECEMBER 2004 䡠 VOLUME 104, NUMBER 13
BLOOD, 15 DECEMBER 2004 䡠 VOLUME 104, NUMBER 13
EXPRESSION OF MOUSE HEMOJUVELIN ORTHOLOG
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Table 1. Relative amounts of liver Hamp and Rgmc mRNA after
administration of iron, erythropoietin, or LPS
Hamp mRNA,
% of control
Rgmc mRNA,
% of control
Iron, 1 wk
484 ⫾ 26*
87 ⫾ 16
Iron, 3 wks
408 ⫾ 91*
79 ⫾ 48
3 ⫾ 2*
81 ⫾ 19
LPS, 90 min
166 ⫾ 66
53 ⫾ 20
LPS, 6 h
128 ⫾ 25
1 ⫾ 1*
Treatment
Erythropoietin
Figure 1. Levels of hepatic Hamp and Rgmc mRNA during prenatal and
postnatal liver development. Liver samples were obtained at days 12.5, 14.5, and
17.5 of embryonic development (E12-E17), within 8 hours after birth (P0), during
postnatal developmental days (P3-P24), or from adult mice aged 2 to 3 months.
Values represent the amount of target mRNA compared to ␤-actin mRNA (means ⫾
SD; n ⫽ 3).
Results and discussion
Real-time PCR allowed detection of Hamp and Rgmc mRNAs in
adult as well as in fetal liver samples, with the amount of Hamp
mRNA exceeding Rgmc mRNA in adult liver by more than 1 order
of magnitude. Tissue-specific expression of Rgmc agreed with
published data2 for human HJV (results not shown).
Hepcidin expression increases during iron overload9 and decreases following erythropoietin administration.10 Subcutaneous
injection of a single dose of iron (600 mg/kg) increased the amount
of Hamp mRNA more than 4-fold when measured 1 week or 3
weeks after treatment; however, the amount of hepatic Rgmc
mRNA was not significantly changed (Table 1). Administration of
erythropoietin for 4 days decreased Hamp mRNA levels to less
than 5% of control values, again without a statistically significant
effect on hepatic Rgmc mRNA levels. These results indicate that, in
contrast to Hamp mRNA, Rgmc mRNA content is not influenced
by iron overload or increased erythropoiesis.
It has been previously shown that HJV is expressed in fetal
liver.2 Because Hamp expression displays significant changes
during both prenatal and postnatal periods,11 we examined whether
the expression pattern of Hamp and Rgmc would be similar.
Although both Hamp and Rgmc mRNAs increased during embryonic liver development, a striking difference was noted in the
postnatal expression of the 2 genes (Figure 1). Hamp mRNA
dropped by 4 orders of magnitude after birth and remained low
until weaning, whereas Rgmc mRNA levels decreased only to about
30% at postnatal day 3 and reached adult levels at day 8. These
results show that the 2 genes are regulated differently during the
postnatal period.
In addition to iron homeostasis, expression of hepcidin is also
regulated by inflammatory cytokines.12,13 Hepcidin was originally
described as an antimicrobial peptide,14 and the link between
hepcidin and the immune response has been further strengthened
by the observations that urinary hepcidin levels rise by 2 orders of
magnitude in patients with infections.2,12 Human hepcidin has
Iron was administered as a single 600-mg/kg dose 1 or 3 weeks prior to death,
erythropoietin as a 50-U daily dose for 4 days preceding the day of death, and LPS as
a single 1-mg/kg dose 90 minutes or 6 hours prior to death. Data are expressed as
means ⫾ SD (n ⫽ 4).
*Statistically significant compared to control group (P ⬍ .05).
therefore been characterized as an acute-phase protein,12 whose
induction is probably responsible for the changes in iron homeostasis during anemia of inflammation. Accordingly, an increase of
hepatic Hamp mRNA has been documented in experimental
animals treated with LPS.9,13,15 As shown in Table 1, a single
injection of LPS slightly increased hepatic Hamp mRNA levels,
measured 6 hours after LPS administration, while decreasing
hepatic Rgmc mRNA levels by more than 1 order of magnitude.
Thus, the response of Hamp and Rgmc to inflammatory stimuli
appears to be fundamentally different.
The link between iron metabolism and inflammation has been
well established, with expression of many of the proteins involved
in iron metabolism responding to infection or LPS treatment.16-18
LPS treatment decreases plasma iron concentrations and generally
down-regulates iron export from the cells. In this respect, it is
interesting to note that the response of Rgmc to LPS resembles the
response of the Slc40a1 gene,18,19 which encodes the iron exporter
ferroportin1. Both hepatic Rgmc and Slc40a1 mRNAs show a
similar decrease following administration of LPS to mice, with
only slight changes at 90 minutes and substantial down-regulation
6 hours after LPS administration.
In conclusion, this study shows that, despite the postulated
functional link between hepcidin and hemojuvelin,2,20 murine
Hamp and Rgmc genes respond differently to changes in iron status
or inflammation. Although the results are based on mRNA
quantification only, and as such do not reflect possible posttranscriptional regulation, they nevertheless indicate that whereas Hamp
mRNA sensitively reacts to iron overload or increased erythropoiesis, hepatic Rgmc mRNA content is not significantly changed. In
addition, hepatic Hamp and Rgmc mRNA levels respond in an
opposite manner to bacterial LPS challenge. The decrease of
hepatic Rgmc mRNA level following LPS treatment suggests that
human HJV expression could be down-regulated during
inflammation.
Acknowledgments
The technical assistance of Dana Duricová and Lydie Tauchenová
is gratefully acknowledged.
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